Steviol Glycosides Content Research Articles

Abiotic stress of ZnO‐PEG, ZnO‐PVP, CuO‐PEG and CuO‐PVP nanoparticles enhance growth, sweetener compounds and antioxidant activities in shoots of Stevia rebaudiana Bertoni

Nanoparticles are known to play remarkable role as abiotic stress elicitors in plants. This study reports the comparative analysis of effects produced by capped [zinc oxide (ZnO)-polyethylene glycol (PEG), ZnO-polyvinyl pyrrolidone (PVP), copper oxide (CuO)-PEG, CuO-PVP] and uncapped (ZnO and CuO) nanoparticles on the medicinal plant, Stevia rebaudiana raised in vitro for the production of commercially important sweetener compounds. In context of shoot organogenesis, ZnO-PEG, ZnO-PVP, CuO-PEG, CuO-PVP were employed to the growth medium that resulted in increased growth parameters, and larger content of steviol glycosides as compared to the shoots raised in medium containing ZnO and CuO, revealed by high-performance liquid chromatography. In the meanwhile, non-enzymatic antioxidant activities including total phenolic content, total flavonoid content, total antioxidant capacity, total reducing power and 2,2-diphenyl-1-picryl hydrazyl-free radical scavenging activity were calculated and showed comparatively greater amounts in shoots grown in medium containing capped ZnO or CuO nanoparticles. Furthermore, the ZnO and its derivatives revealed to be more reactive at 1 mg/l of concentration. Whereas, the CuO and its derivatives produced greater response on Stevia at 10 mg/l concentration of nanoparticles. This study paves the way for more such studies encompassing capped and uncapped nanoparticles and their ultimate effect on in-vitro grown plant tissues for the production of active metabolites on industrial scale.

Stevia rebaudiana Bertoni: A Natural Alternative for Treating Diseases Associated with Metabolic Syndrome.

Stevia rebaudiana (SR) is often used by the food industry due to its steviol glycoside content, which is a suitable calorie-free sweetener. Further, both in vitro and in vivo studies indicate that these glycosides and the extracts from SR have pharmacological and therapeutic properties, including antioxidant, antimicrobial, antihypertensive, antidiabetic, and anticancer. This work reviews the antiobesity, antihyperglycemic, antihypertensive, and antihyperlipidemic effects of the majority of glycosides and aqueous/alcoholic extracts from the leaves, flowers, and roots of the SR. These compounds can serve as a natural and alternative treatment for diseases that are associated with metabolic syndrome, thus contributing to health promotion.

Comparison of different in vitro micropropagation methods of Stevia rebaudiana B. including temporary immersion bioreactor (BIT®)

Stevia rebaudiana is beneficial to treat diabetes because of its low-calorie glucoside sweeteners. Natural and vegetative propagation are inefficient. In vitro techniques are an attractive alternative but low propagation and reproducibility rates have been reported. Therefore, different ways to increase natural sweetener production in vitro, such as BIT®, are required. We compared semisolid medium, liquid medium and BIT® in terms of Stevia biomass and steviol glycosides production. At 21 days of culture, morphological quality of BIT®-derived shoots was best and coupled with shoot fresh and dry weight that were more than seven times higher in BIT® compared with micropropagation in liquid or semisolid media. In turn, the total content of steviol glycosides produced was also higher in bioreactors. The usefulness of BIT® to produce plant metabolites in vitro is again demonstrated, even if additional experiments are required to increase the economic efficiency of the process.

Effects of light intensity and photoperiod on improving steviol glycosides content in Stevia rebaudiana (Bertoni) Bertoni while conserving light energy consumption

Stevia rebaudiana (Bertoni) Bertoni accumulates steviol glycosides (SGs) which are natural sweeteners. Leaf extracts have been widely used as a food additive because the strong sweetness intensity of SGs that is 300 times sweeter than sucrose. S. rebaudiana is a short-day (SD) perennial plant, and some studies claim that long-day (LD) treatment affects SGs accumulation. However, the optimal and detailed light regime required to increase total SGs content remains controversial. We analyzed the effect of various light intensities and day lengths to determine optimal light treatment for SGs accumulation with minimal energy consumption in growth chambers. Night-interruption (NI) treatments in conjunction with 8h photoperiod increased leaf biomass to amounts observed under LD conditions, and resulted in higher SGs content than that observed under 12h photoperiod while minimizing energy consumption. In addition, end-of-day far-red (EOD-FR) treatment for 15min at 50μmolm−2s−1 was associated with transcription upregulation of the SGs-related gene UGT85C2, higher SGs content, and similar leaf biomass compared to that observed under 16h photoperiod. These light treatments offer great potential for energy-efficient increases in S. rebaudiana total SGs content when plants are grown in a greenhouse or in the field with supplemental lights.

Salt-tolerance mechanisms induced in Stevia rebaudiana Bertoni: Effects on mineral nutrition, antioxidative metabolism and steviol glycoside content

In order to cope with challenges linked to climate change such as salinity, plants must develop a wide spectrum of physiological and molecular mechanisms to rapidly adapt. Stevia rebaudiana Bertoni plants are a case in point. According to our findings, salt stress has no significant effect on plant growth in these plants, which accumulate sodium (Na+) in their roots, thus avoiding excessive Na+ accumulation in leaves. Furthermore, salt stress (NaCl stress) increases the potassium (K+), calcium (Ca2+), chloride ion (Cl−) and proline concentrations in Stevia leaves, which could contribute to osmotic adjustment. We also found that long-term NaCl stress does not produce changes in chlorophyll concentrations in Stevia leaves, reflecting a mechanism to protect the photosynthesis process. Interestingly, an increase in chlorophyll b (Chlb) content occured in the oldest plants studied. In addition, we found that NaCl induced reactive oxygen species (ROS) accumulation in Stevia leaves and that this accumulation was more evident in the presence of 5 g/L NaCl, the highest concentration used in the study. Nevertheless, Stevia plants are able to induce (16 d) or maintain (25 d) antioxidant enzymes to cope with NaCl-induced oxidative stress. Low salt levels did not affect steviolbioside and rebaudioside A contents. Our results suggest that Stevia plants induce tolerance mechanisms in order to minimize the deleterious effects of salt stress. We can thus conclude that saline waters can be used to grow Stevia plants and for Steviol glycosides (SGs) production.

Impact of blue, red, and far-red light treatments on gene expression and steviol glycoside accumulation in Stevia rebaudiana

Stevia rebaudiana (Bertoni) Bertoni is a plant that biosynthesizes a group of natural sweeteners that are up to approximately 400 times sweeter than sucrose. The sweetening components of S. rebaudiana are steviol glycosides (SGs) that partially share their biosynthesis pathway with gibberellins (GAs). However, the molecular mechanisms through which SGs levels can be improved have not been studied. Therefore, transcription levels of several SG biosynthesis-related genes were analyzed under several light treatments involved in GA biosynthesis. We detected higher transcription of UGT85C2, which is one of the UDP-glycosyltransferases (UGTs) involved in catalyzing the sugar-transfer reaction, under red/far-red (R/FR) 1.22 light-emitting diodes (LEDs) and blue LEDs treatment. In this study, it was demonstrated that transcription levels of SG-related genes and the SGs content are affected by light treatments known to affect the GA contents. It is expected that this approach could serve as a practical way to increase SG contents using specific light treatments.

A new natural source for obtainment of inulin and fructo-oligosaccharides from industrial waste of Stevia rebaudiana Bertoni.

Fructan-type inulin and fructo-oligosaccharides (FOS) are reserve polysaccharides that offer an interesting combination of nutritional and technological properties for food industry. Stevia rebaudiana is used commercially in the sweetener industry due to the high content of steviol glycosides in its leaves. With the proposal of using industrial waste, the objective of the present study was to isolate, characterize and evaluate the prebiotic activity of inulin and FOS from S. rebaudiana stems. The chemical characterization of the samples by GC-MS, NMR and off-line ESI-MS showed that it was possible to obtain inulin molecules from the S. rebaudiana stems with a degree of polymerization (DP) of 12, and FOS with a DP<6. The in vitro prebiotic assay of these molecules indicates a strain specificity in fermentation capacity of fructans as substrate. FOS molecules with a low DP are preferably fermented by beneficial microbiota than inulin molecules with higher DP.

Soil Water Tension Irrigation Criteria Affects Stevia rebaudiana Leaf Yield and Leaf Steviol Glycoside Composition

Stevia (Stevia rebaudiana Bertoni) is of interest for the production of steviol glycosides due to their noncaloric sweetening properties. Commercial stevia leaf production to date has focused on rebaudioside A and stevioside. Relatively little is known about the cultural practices necessary for the efficient production of stevia leaf and steviol glycosides. Irrigation management is an important agronomic technique that growers can use to obtain high yield while also pursuing water use efficiency (WUE). This trial investigated the effect that irrigation onset criteria measured as soil water tension (SWT) had on dry stevia leaf yields, steviol glycoside content and yield, and steviol glycoside ratios. Two stevia cultivars, SW 107 and SW 129 (S&amp;W Seed Company Inc., Fresno, CA), were subjected to SWT irrigation criteria of 10, 20, 40, 60, and 80 kPa over a 57-day trial period at the Oregon State University (OSU) Malheur Experiment Station, Ontario, OR. Harvested plant material was evaluated for dry leaf yield and content of several steviol glycosides. Analysis of variance (ANOVA) showed significant cultivar differences in leaf yield, leaf stevioside and rebaudioside A content and yield, and steviol glycoside ratios (P = 0.05). Examining the data by ANOVA, dry leaf yield, stevioside percent, rebaudioside C percent, total steviol glycoside (TSG) percent, rebaudioside C yield, and TSG yield were significantly greater among the wetter (closer to 10 kPa) compared with the drier (closer to 80 kPa) SWT criteria treatments, yet a preferred treatment regime was not clearly evident. When comparing cultivars by regression analysis, there was a highly significant decrease in the stevioside content, and an increase in the ratio of rebaudioside A to stevioside from wetter to drier irrigation onset criteria (due to lower stevioside), whereas the rebaudioside A content did not respond significantly to differing irrigation criteria. Increasing dry leaf productivity with wetter irrigation criteria (closer to 10 kPa), directly increased the total rebaudioside A yield also, which in turn could provide increased crop value to the grower. Irrigating near 10 kPa produced higher dry leaf yield and total rebaudioside A yield, than irrigating at drier criteria.

Endophytic root fungus Piriformospora indica affects transcription of steviol biosynthesis genes and enhances production of steviol glycosides in Stevia rebaudiana

Stevia rebaudiana (Bertoni) produces low calorie sweeteners, steviol glycosides (SGs) - stevioside and rebaudioside-A. Varying spore concentrations of P. indica were applied to S. rebaudiana grown in vitro and in the greenhouse to examine the effect on plant growth and synthesis of SGs. Symbiotic association of P. indica showed plant growth promotion and higher SGs content. The transcription profiles of eight key genes of the SGs biosynthetic pathway also showed strong upregulation. The results show that the symbiotic effect of P. indica can be utilized to achieve plant growth enhancement in S. rebaudiana along with increased production of SGs.

Dry Leaf and Steviol Glycoside Productivity of Stevia rebaudiana in the Western United States

Stevia (Stevia rebaudiana Bertoni) is a perennial herbaceous plant native to Paraguay, where it was used by the native Guarani peoples for centuries. Although steviol glycosides from stevia are powerful natural noncaloric sweeteners, stevia has been cultivated and commercially available only for the past 50 years. Cultural practices are still in development, and productivity potential in the United States is largely unknown. Currently commercial growers and processors worldwide are seeking to maximize the productivity of rebaudioside A, a steviol glycoside. The trials reported here examined the effects of location, harvest strategy, and cultivar on stevia dry leaf yield, steviol glycoside content, and steviol glycoside yield. Six or seven stevia cultivars were grown for ≈5 months at four western U.S. locations, with an irrigation criterion of 20 kPa. Stevia at every location was subjected to two harvest strategies: either one harvest at the end of the season or two harvests, one midseason and another at the end of the season. The main plots at each location were the stevia cultivars, and the split plots were the harvest strategies in a randomized complete block, split-plot design with four replicates. Dry leaf yield, leaf steviol glycoside content, and leaf steviol glycoside yield varied by cultivar, location, and cultivar by location, but not by harvest strategy or interactions of harvest strategy with location or cultivar. Dry leaf yield averaged 4.12 Mg·ha−1 with significant differences by cultivar and location. One of the steviol glycosides, rebaudioside A yield averaged 300 kg·ha−1 with significant differences by cultivar and by interactions of location with cultivar. Leaf productivity was greater at Ontario, OR, than at Hanford, CA, Indio, CA, or Yuma, AZ. Dry leaf yield greater than 7 Mg·ha−1 and rebaudioside A yields greater than 500 kg·ha−1 were observed at Ontario. Stevia perenniated at Hanford and Indio, providing the option of multiyear harvests from a single planting.

Gene transcription and steviol glycoside accumulation in Stevia rebaudiana under polyethylene glycol-induced drought stress in greenhouse cultivation.

Stevia rebaudiana is a sweet herb of the Astraceae family, which is cultivated for the natural sweeteners it contains. The aim of this study was to assess the effect of drought, simulated by the application of polyethylene glycol (5%, 10%, and 15% w/v), on the content of steviol glycosides (SVglys) and transcription levels of six genes involved in the biosynthesis of these natural sweeteners. The transcription levels of ent‐kaurene synthase, ent‐kaurene oxidase, ent‐kaurenoic acid hydroxylase, and three UDP‐dependent glycosyltransferases, UGT85C2,UGT74G1 and UGT76G1 were downregulated under polyethylene glycol treatment. Polyethylene glycol treatment significantly decreased the amount of stevioside, rebaudioside A, B, C and F, steviolbioside, dulcoside A, rubusoside, and total SVglys. These results strongly suggest a close relationship of SVglys content with the transcription of genes involved in the SVglys biosynthesis pathway. Comparing the observations of the present study with other reports provided the knowledge that the Stevia response to drought stress can be influenced by different environmental and experimental factors, in addition to intensity of drought stress. In conclusion, these results strongly suggest that polyethylene glycol‐induced drought stress has a negative effect on the content of SVglys and transcription of SVglys biosynthetic genes and that this should be investigated further. We recommend that sufficient irrigation of Stevia is required to obtain a high content of SVglys.

An efficient regeneration system and steviol glycoside analysis of Stevia rebaudiana Bertoni, a source of natural high-intensity sweetener

Stevia is a natural, zero-calorie, intensively sweet extracted from the leaves of Stevia rebaudiana. Its sweet taste derives from a group of compounds known as steviol glycosides. In this study, an efficient micropropagation protocol for S. rebaudiana was developed for possible commercial implementation using Murashige and Skoog (MS) basal medium without plant growth regulators for most of the process. Direct shoot formation was achieved after cultivation of nodal segments on MS medium with or without plant growth regulators (benzyl amino purine [BAP], kinetin [KIN]) at various concentrations (0.1, 0.5, 1.0, or 2.0 mg L−1). Although all treatments produced two shoots per explants after 3 wk of culture, high concentrations of KIN and BAP (1.0 or 2.0 mg L−1) induced more callus formation. Rooting was achieved on MS medium containing 0.25 mg L−1 indole-3-acetic acid (IAA), which produced 8.1 roots per shoot after 3 wk of cultivation. After acclimatization of the regenerants in a portable greenhouse for 3 wk, all regenerants and seed-derived seedlings were transferred to field conditions for 16 wk. Steviol glycoside contents (% leaf dry weight) did not differ between leaves collected from regenerants and seed-derived plants. Rebaudioside A content ranged from 4.7 to 5.0% (w/w), while stevioside ranged from 6.4 to 6.9% (w/w). There was no significant difference between the two sampling periods (late vegetative and flowering stages) for the plants grown in the field. In this study, a cost-effective in vitro regeneration protocol was established that enables efficient, large-scale in vitro production of S. rebaudiana for field cultivation.

Effects of growth stage and fulvic acid on the diversity and dynamics of endophytic bacterial community in Stevia rebaudiana Bertoni leaves.

The aim of this study was to learn the interactions among the endophytic bacteria, the plant growth, the foliar spray of fulvic acid, and the accumulation of steviol glycosides in the leaves of Stevia rebaudiana. Metagenomic DNA was extracted from the Stevia leaves at different growth stages with or without the fulvic acid treatment; and the diversity of endophytic bacteria in Stevia leaves was estimated by pyrosequencing of 16S rRNA genes. As results, Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes were found to be the dominant phyla despite the growth stages and fulvic acid application. Stevia growth stages strongly regulated composition of endophytic community. The genera Agrobacterium (12.3%) and Erwinia (7.2%) dominated in seedling stage were apparently declined in the vegetable and initial flowering stages, while Sphingomonas and Methylobacterium increased in mature leaves at harvest time, which showed that the mature leaves of Stevia preferred to accumulate some certain endophytic bacteria. Sphingomonas and Methylobacterium constituted an important part of the core endophytic community and were positively correlated with the stevioside content and UGT74G1 gene expression, respectively; while Erwinia, Agrobacterium, and Bacillus were negatively correlated with the stevioside accumulation. Fulvic acid treatment accelerated the variation of endophytes along the growth stages and increased the steviol glycosides content. This is the first study to reveal the community composition of endophytic bacteria in the Stevia leaves, to evidence the strong effects of growth stage and fulvic acid application on the endophytes of Stevia, and to demonstrate the correlation between the endophytic bacteria and the steviol glycosides accumulation.

Combined effects of Piriformospora indica and Azotobacter chroococcum enhance plant growth, antioxidant potential and steviol glycoside content in Stevia rebaudiana

The herb Stevia rebaudiana Bertoni is an important sugar substitute due to the presence of steviol glycosides-mainly stevioside and rebaudioside-A. These compounds possess antioxidant activity with industrial importance. The fungus Piriformospora indica has been used to improve medicinally important biomolecules, growth and secondary metabolite content in plants. The soil bacterium Azotobacter chroococcum has also been reported to possess plant growth promoting ability. The present study was undertaken to test the ability of P. indica (Pi) and A. chroococcum (Az), alone and in combination to improve the growth, antioxidant activity and steviol glycoside (SG) content of in vitro S. rebaudiana plantlets. Inoculation with Pi or Az alone showed enhancement in all these characters compared with non-inoculated plantlets. Combined inoculation with Pi and Az further enhanced these traits.

Towards a semi-perennial culture of Stevia rebaudiana (Bertoni) Bertoni under temperate climate: effects of genotype, environment and plant age on steviol glycoside content and composition

Stevia rebaudiana (Bertoni) Bertoni is a perennial shrub native to Paraguay whose leaves accumulate sweetening compounds of high value: steviol glycosides (SVglys). These diterpenoids differ in the number (1–4) and the nature of sugar units bound to a steviol skeleton: mainly glucose but also rhamnose and xylose. The two main SVglys are stevioside (ST; 3 glucose units) and rebaudioside A (RA, 4 glucose units). Along with an evaluation of genotypic variability for SVglys content and composition, this study investigates the winter survival and the effect of perennial cultivation on S. rebaudiana genotypes through a 2-year experiment conducted on two locations. The first year, a population of 96 genotypes was examined in a nursery field (site A) and a subset of five genotypes was grown in a field trial (site B). The winter surviving genotypes (16 on site A and 5 on site B) were studied the following year, alongside new plantings of the same lines (8 on site A and 5 on site B). The genotypes showed a high variability in SVgly content (from 45.7 to 186.2 mg g−1 DM) and composition along four distinct clusters. There was no correlation between total SVglys content and composition. Genotypes without RA had no other SVglys with more than three conjugated sugars. SVgly content varied among environments and generally increased between 1- and 2-year-old plants, while SVgly composition remained stable. This study showed that breeding for winter survival in temperate conditions would allow an increase in SVgly productivity in a semi-perennial stevia production.

In silico analysis and expression profiling of miRNAs targeting genes of steviol glycosides biosynthetic pathway and their relationship with steviol glycosides content in different tissues of Stevia rebaudiana

miRNAs are emerging as potential regulators of the gene expression. Their proven promising role in regulating biosynthetic pathways related gene networks may hold the key to understand the genetic regulation of these pathways which may assist in selection and manipulation to get high performing plant genotypes with better secondary metabolites yields and increased biomass. miRNAs associated with genes of steviol glycosides biosynthetic pathway, however, have not been identified so far. In this study miRNAs targeting genes of steviol glycosides biosynthetic pathway were identified for the first time whose precursors were potentially generated from ESTs and nucleotide sequences of Stevia rebaudiana. Thereafter, stem-loop coupled real time PCR based expressions of these miRNAs in different tissues of Stevia rebaudiana were investigated and their relationship pattern was analysed with the expression levels of their target mRNAs as well as steviol glycoside contents. All the miRNAs investigated showed differential expressions in all the three tissues studied, viz. leaves, flowers and stems. Out of the eleven miRNAs validated, the expression levels of nine miRNAs (miR319a, miR319b, miR319c, miR319d, miR319e, miR319f, miR319h, miRstv_7, miRstv_9) were found to be inversely related, while expression levels of the two, i.e. miR319g and miRstv_11 on the contrary, showed direct relation with the expression levels of their target mRNAs and steviol glycoside contents in the leaves, flowers and stems. This study provides a platform for better understanding of the steviol glycosides biosynthetic pathway and these miRNAs can further be employed to manipulate the biosynthesis of these metabolites to enhance their contents and yield in S. rebaudiana.

Biomass Yield and Steviol Glycoside Production in Callus and Suspension Culture of Stevia rebaudiana Treated with Proline and Polyethylene Glycol.

Enhanced production of steviol glycosides (SGs) was observed in callus and suspension culture of Stevia rebaudiana treated with proline and polyethylene glycol (PEG). To study their effect, yellow-green and compact calli obtained from in vitro raised Stevia leaves were sub-cultured on MS medium supplemented with 2.0mg l(-1) NAA and different concentrations of proline (2.5-10mM) and PEG (2.5-10%) for 2weeks, and incubated at 24 ± 1°C and 22.4μmol m(-2) s(-1) light intensity provided by white fluorescent tubes for 16h. Callus and suspension culture biomass (i.e. both fresh and dry weight content) was increased with 5mM proline and 5% PEG, while at further higher concentrations, they got reduced. Further, quantification of SGs content in callus (collected at 15th day) and suspension culture (collected at 10th and 15th day) treated with and without elicitors was analysed by HPLC. It was observed that chemical stress enhanced the production of SGs significantly. In callus, the content of SGs increased from 0.27 (control) to 1.09 and 1.83% with 7.5mM proline and 5% PEG, respectively, which was about 4.0 and 7.0 times higher than control. However, in the case of suspension culture, the same concentrations of proline and polyethylene glycol enhanced the SG content from 1.36 (control) to 5.03 and 6.38%, respectively, on 10th day which were 3.7 times and 4.7 times higher than control.

In silico analysis, expression profiling and correlation of miRNAs targeting genes of steviol glycosides biosynthesis with steviol glycosides contents in different tissues of Stevia rebaudiana

In silico analysis, expression profiling and correlation of miRNAs targeting genes of steviol glycosides biosynthesis with steviol glycosides contents in different tissues of Stevia rebaudiana

Optimization of Ultrasound Assisted Extraction of Functional Ingredients from Stevia Rebaudiana Bertoni Leaves

Abstract The aim of the present study was to reveal an effective extraction procedure for maximization of the yield of steviol glycosides and total phenolic compounds as well as antioxidant activity in stevia extracts. Ultrasound assisted extraction was compared with conventional solvent extraction. The examined solvents were water (100°C/24 h) and 70% ethanol (at 70°C for 30 min). Qualitative and quantitative analyses of steviol glycosides in the extracts obtained were performed using high performance liquid chromatography. Total phenolic compounds, flavonoids, and radical scavenging capacity by 2, 2-azino-di-3-ethylbenzothialozine- sulphonic acid) assay were also determined. The highest content of steviol glycosides, total phenolic compounds, and flavonoids in stevia extracts were obtained when ultrasound assisted extraction was used. The antioxidant activity of the extracts was correlated with the total amount of phenolic compounds. The results indicated that the examined sonication parameters represented as the probe diameter (7 and 22 mm) and treatment time (2, 4, 6, 8, and 10 min) significantly contributed to the yield of steviol glycosides, total phenolic compounds, and flavonoids. The optimum conditions for the maximum yield of steviol glycosides, total phenolic compounds, and flavonoids were as follows: extraction time 10 min, probe diameter 22 mm, and temperature 81.2°C.

Impact of nitrogen supply on growth, steviol glycosides and photosynthesis in Stevia rebaudiana Bertoni

This work investigated the agronomic, physiological and biochemical response of Stevia rebaudiana Bertoni grown under different nitrogen (N) rates. A pot trial in open air conditions was set up in 2012 with the aim to evaluate the effect of four N rates on the biometric and productive characteristics, steviol glycoside (SG) content as well as on leaf gas exchanges, chlorophyll fluorescence, photosynthetic pigments, Rubisco activity and N use efficiency. N deficiency caused a decrease in leaf N content, chlorophylls and photosynthetic CO2 assimilation, resulting in a lower dry matter accumulation as well as in reduced SG production. The application of 150 kg N ha− 1 seems to be the most effective treatment to improve rebaudioside A (Reb A) content, Reb A/stevioside ratio, photosynthetic CO2 assimilation, stomatal conductance, N use efficiency, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) and PSII efficiency. The results demonstrate that by using an appropriate N rate it is possible to modulate the SG biosynthesis, with a significant increase in the Reb A content and in the ratio between Reb A and stevioside. This finding is of great relevance in order to obtain a raw material designed to meet consumer needs and bio-industry requirements for high-quality, Reb A content, and safe and environmentally friendly products.